Type III Secretion System Targeting Molecules

ABSTRACT

This invention relates generally to molecules that specifically bind bacterial V-tip proteins of the type III secretion system of Gram negative bacteria such as PcrV from  Pseudomonas aeruginosa . More specifically, this invention relates to molecules that block the injection of effector molecules into target cells. This invention also relates to molecules that specifically bind to bacterial lipoproteins, such as OprI. The molecules of the present invention are monospecific or multispecific and can bind their target antigen in a monovalent or multivalent manner. The invention also relates generally to molecules that specifically bind bacterial cell surface proteins such as OprI, and to methods of use these molecules in a variety of therapeutic, diagnostic, and/or prophylactic indications.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/144,470, filed May 2, 2016, which claims the benefit of U.S.Provisional Application No. 62/155,967, filed May 1, 2015, and U.S.Provisional Application No. 62/254,992, filed Nov. 13, 2015; thecontents of each of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates generally to molecules that specifically bindbacterial V-tip proteins of the type III secretion system of Gramnegative bacteria such as PcrV from Pseudomonas aeruginosa. Morespecifically, this invention relates to molecules that block orotherwise inhibit the injection of effector molecules into target cells.The molecules of the present invention are monospecific or multispecificand can bind their target antigen(s) in a monovalent or multivalentmanner. In some embodiments, these antibodies may be combined with abacterial surface targeting component, binding to the Pseudomonasprotein, OprI. The invention also relates generally to molecules thatspecifically bind bacterial cell surface proteins such as OprI, and tomethods of use these molecules in a variety of therapeutic, diagnostic,and/or prophylactic indications.

BACKGROUND OF THE INVENTION

V-tip proteins of Pseudomonas aeruginosa (PcrV) are essential componentsof the bacterial type III secretion system (T3SS) that is capable ofinjecting toxic effector molecules into eukaryotic cells. The V-tipproteins are localized at the extreme end of the T3SS apparatus andoligomerization is thought to be necessary for the functionaltranslocalization of the effector molecules across target cellmembranes.

OprI is an outer membrane lipoprotein in Pseudomonas aeruginosa andother Pseudomonas species. OprI is highly conserved in P. aeruginosastrains and therefore represents an excellent candidate for cell-surfacetargeting of Pseudomonas bacteria.

Accordingly, there exists a need for compositions and therapies thattarget V-tip proteins of Gram negative bacteria and cell-surfacecomponents such as OprI.

SUMMARY OF THE INVENTION

The disclosure provides molecules, e.g., polypeptides includingantibodies, antigen-binding antibody fragments, antibody-likepolypeptides, and/or fusion polypeptides, and compositions that bindbacterial V-tip proteins of the type III secretion system of Gramnegative bacteria, such as PcrV from Pseudomonas aeruginosa, and/orcell-surface proteins such as the OprI protein of Pseudomonasaeruginosa, and methods of making and using these compositions in avariety of therapeutic, diagnostic, and/or prophylactic indications. TheV-tip protein targeting molecules of the present invention aremonospecific or multispecific and can bind their target antigen(s) in amonovalent or multivalent manner.

These molecules are useful in binding and neutralizing or otherwiseinhibiting at least one biological activity of one or more bacterialV-tip proteins of the type III secretion system of Gram negativebacteria.

Pseudomonas aeruginosa and other drug resistant Gram negative bacteriaare a major health concern, causing community acquired and nosocomialinfections. Infections with such bacteria can be serious andlife-threatening. An important virulence factor is the type 3 secretionsystem (T3 SS). The T3 SS of Gram negative bacteria is responsible fortranslocation of toxins into eukaryotic cells, causing cell death andlysis, thereby allowing the bacterium to establish infection.

PcrV of Pseudomonas aeruginosa, is an example of a V-tip protein commonto many Gram negative bacterial T3 SSs. The V-tip protein is located atthe extreme end of the T3 SS apparatus and oligomerization is thought tobe necessary for the functional translocalization of the effectormolecules across target cell membranes. PcrV of P. aeruginosa isrequired for injection of effector molecules (ExoS, ExoT, ExoU, andExoY) into the eukaryotic cell cytosol, resulting in cell death andlysis. PcrV of P. aeruginosa has been shown to be a protective antigen,suggesting that targeting the V-tip proteins of numerous Gram negativebacteria will provide an effective therapeutic option.

In some embodiments, the V-tip protein targeting molecules areantibodies and antibody-like molecules that specifically bind Gramnegative bacterial V-tip proteins of the type 3 secretion system (T3SS)apparatus and block the cytotoxicity toward eukaryotic cells. In someembodiments, the V-tip protein targeting antibody, referred to as theV-tip protein binding proteins (VPBP) are derived from antibodies orantigen-binding antibody fragments including, for example, single-chainvariable fragments (scFv), Fab fragments, single domain antibodies(sdAb), V_(NAR), or VHHs. In preferred embodiments, the VPBPs are humanor humanized sdAb. The sdAb fragments can be derived from VHH, V_(NAR),engineered VH or VK domains. VHHs can be generated from camelid heavychain only antibodies. VNARS can be generated from cartilaginous fishheavy chain only antibodies. Various methods have been implemented togenerate monomeric sdAbs from conventionally heterodimeric VH and VKdomains, including interface engineering and selection of specificgermline families.

In other embodiments, the VPBPs are derived from non-antibody scaffoldproteins for example but not limited to designed ankyrin repeat proteins(darpins), avimer, anticalin/lipocalins, centyrins and fynomers.

In preferred embodiments, the V-tip protein is or is derived from thePseudomonas aeruginosa PcrV, and the VPBP specifically binds PcrV. Insome embodiments, the VPBP is able to bind 2 or more VPBPs of variousGram negative bacteria, including at least PcrV from Pseudomonasaeruginosa. In some embodiments, the VPBP binds to a V-tip protein froma single Gram negative bacterial species such as PcrV from Pseudomonasaeruginosa. In some embodiments, the VPBP binds to a V-tip protein frommore than one Gram negative bacterial species, including at least PcrVfrom Pseudomonas aeruginosa, and is thereby considered speciescross-reactive.

In some embodiments, the V-tip protein targeting molecule is a fusionprotein. Unexpectedly, it was discovered that enhancing the valency ofthe VPBP greatly enhanced the efficacy of cyto-protection fromPseudomonas aeruginosa both in vitro and in vivo. More surprisingly, itwas found that targeting two distinct epitopes on PcrV with the distinctVPBPs in a single multispecific fusion protein resulted in an evengreater protection from cytotoxicity caused by Pseudomonas aeruginosa.The later finding held true, even when the individual monospecific VPBPwas only weakly protective. In fact, it was found that when VPBPrecognizing distinct epitopes on PCRV were incorporated into a singlefusion protein, they were more potent at cyto-protection compared toVPBP-containing fusion proteins that were multivalent to the sameepitope on PCRV or to the combination to two separate monospecificVPBP-containing fusion proteins each recognizing a distinct epitope onPCRV.

In some embodiments, the present invention includes fusion proteinsincorporating more than one VPBP and are referred to herein asmultivalent. In some embodiments, the VPBPs of the fusion proteinrecognize the same epitope on the target V-protein and are referred toherein as monospecific-multivalent. In other embodiments, the VPBPs ofthe fusion protein recognize distinct epitopes on the target V-proteinand are referred to herein as multispecific-multivalent. In someembodiments, the VPBP-containing fusion protein includes two VPBPs andhas a bivalent binding capacity toward the target V-tip protein. In someembodiments, the VPBP-containing fusion protein includes three VPBPs andhas a trivalent binding capacity toward the target V-tip protein. Insome embodiments, the VPBP-containing fusion protein includes four VPBPsand has a tetravalent binding capacity toward the target V-tip protein.In some embodiments, the VPBP-containing fusion protein includes sixVPBPs and has a hexavalent binding capacity toward the target V-tipprotein. In some embodiments, the VPBP-containing fusion proteinincludes eight VPBPs and has an octavalent binding capacity toward thetarget V-tip protein. In these embodiments, the VPBPs incorporated intothe fusion protein of the present invention can be monospecific ormultispecific.

Generally the fusion proteins of the present invention consist of atleast two or more VPBPs operably linked via a linker polypeptide. Theutilization of sdAb fragments as the specific VPBP within the fusion thepresent invention has the benefit of avoiding the heavy chain: lightchain mis-pairing problem common to many bi/multispecific antibodyapproaches. In addition, the fusion proteins of the present inventionavoid the use of long linkers necessitated by many bispecificantibodies. Furthermore, the fusion proteins of the present inventionare generally smaller in size (ranging approximately from 75 to 125 kDa)than a conventional antibody. This reduced molecular weight maybe enablebetter penetration into site of infection compared to conventionalantibodies.

In some embodiments, the fusion protein of the present invention iscomposed of a single polypeptide. In other embodiments, the fusionprotein of the present invention is composed of more than onepolypeptide. For example, wherein a heterodimerization domain isincorporated into the fusion protein so as the construct an asymmetricfusion protein. For example if an immunoglobulin Fc region isincorporated into the fusion protein the CH3 domain can be used ashomodimerization domain, or the CH3 dimer interface region can bemutated so as to enable heterodimerization.

In some embodiments, the fusion protein contains the VPBPs on oppositeends. For example the VPBPs are located on both the amino-terminal(N-terminal) portion of the fusion protein and the carboxy-terminal(C-terminal) portion of the fusion protein. In other embodiments, allthe VPBPs reside on the same end of the fusion protein. For example,VPBPs reside on either the amino or carboxyl terminal portions of thefusion protein.

In some embodiments, the fusion protein lacks an Fc region.

In some embodiments, the fusion protein contains an immunoglobulin Fcregion. In some embodiments, the immunoglobulin Fc region is an IgGisotype selected from the group consisting of IgG1 subclass, IgG2subclass, IgG3 subclass, and IgG4 subclass.

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment thereof is an IgG isotype. For example, theimmunoglobulin Fc region of the fusion protein is of human IgG1subclass, having an amino acid sequence:

(SEQ ID NO: 1)

VFLFPPKPKD  TLMISRTPEV TCVVVDVSHE DPEVKFNWYV  DGVEVHNAKT

YRVVSVLTVL  HQDWLNGKEY KCKVSNKALP APIEKTISKA  KGQPREPQVY TLPPSRDELTKNQVSLTCLV  KGFYPSDIAV EWESNGQPEN NYKTTPPVLD  SDGSFFLYSK LTVDKSRWQQGNVFSCSVMH  EALHNHYTQK SLSLSPGK

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment thereof comprises a human IgG1 polypeptide sequence thatis at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQID NO: 1.

In some embodiments, the human IgG1 Fc region is modified at amino acidAsn297 (Boxed, Kabat Numbering) to prevent glycosylation of the fusionprotein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In someembodiments, the Fc region of the fusion protein is modified at aminoacid Leu235 (Boxed, Kabat Numbering) to alter Fc receptor interactions,e.g., Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, theFc region of the fusion protein is modified at amino acid Leu234 (Boxed,Kabat Numbering) to alter Fc receptor interactions, e.g., Leu234Ala(L234A). In some embodiments, the Fc region of the fusion protein isaltered at both amino acid 234 and 235, e.g., Leu234Ala and Leu235Ala(L234A/L235A) or Leu234Val and Leu235Ala (L234V/L235A). In someembodiments, the Fc region of the fusion protein is lacking an aminoacid at one or more of the following positions to reduce Fc receptorbinding: Glu233 (E233, Bold in SEQ ID NO: 1), Leu234 (L234), or Leu235(L235). In some embodiments, the Fc region of the fusion protein isaltered at Gly235 to reduce Fc receptor binding. For example, whereinGly235 is deleted from the fusion protein. In some embodiments, thehuman IgG1 Fc region is modified at amino acid Gly236 to enhance theinteraction with CD32A, e.g., Gly236Ala (G236A, Boxed in SEQ ID NO: 1).In some embodiments, the human IgG1 Fc region is lacks Lys447, whichcorresponds to residue 218 of SEQ ID NO: 1 (EU index of Kabat et al 1991Sequences of Proteins of Immunological Interest).

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment of the fusion protein is of human IgG2 subclass, havingan amino acid sequence:

(SEQ ID NO: 2) PAPPVAGPSV    FLFPPKPKDT  LMISRTPEVT CVVVDVSHEDPEVQFNWYVD  GVEVHNAKTK

   RVVSVLTVVH  QDWLNGKEYK CKVSNKGLPA PIEKTISKTK  GQPREPQVYT LPPSREEMTKNQVSLTCLVK   GFYPSDISVE WESNGQPENN YKTTPPMLDS  DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE  ALHNHYTQKS LSLSPGK

In some embodiments, the fusion or immunologically active fragmentthereof comprises a human IgG2 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the human IgG2 Fc region is modified at amino acidAsn297 (Boxed, to prevent to glycosylation of the antibody, e.g.,Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the humanIgG2 Fc region lacks Lys447, which corresponds to residue 217 of SEQ IDNO: 2 (EU index of Kabat et al 1991 Sequences of Proteins ofImmunological Interest).

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment of the fusion protein is of human IgG3 subclass, havingan amino acid sequence:

(SEQ ID NO: 3) PAPELLGGPS  VFLFPPKPKD   TLMISRTPEV TCVVVDVSHEDPEVQFKWYV     DGVEVHNAKT

FRVVSVLTVL   HQDWLNGKEY KCKVSNKALP  APIEKTISKT  KGQPREPQVY TLPPSREEMT KNQVSLTCLV  KGFYPSDIAV  EWESSGQPEN NYNTTPPMLD  SDGSFFLYSK LTVDKSRWQQ GNIFSCSVMH 

SLSLSPGK  

In some embodiments, the antibody or immunologically active fragmentthereof comprises a human IgG3 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the human IgG3 Fc region is modified at amino acidAsn297 (Boxed, Kabat Numbering) to prevent to glycosylation of theantibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In someembodiments, the human IgG3 Fc region is modified at amino acid 435 toextend the half-life, e.g., Arg435His (R435H, boxed in SEQ ID NO: 3). Insome embodiments, the human IgG3 Fc region is lacks Lys447, whichcorresponds to residue 218 of SEQ ID NO: 3 (EU index of Kabat et al 1991Sequences of Proteins of Immunological Interest).

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment of the fusion protein is of human IgG4 subclass, havingan amino acid sequence:

(SEQ ID NO: 4)

VFLFPPKPKD   TLMISRTPEV  TCVVVDVSQE DPEVQFNWYV    DGVEVHNAKT

YRVVSVLTVL    HQDWLNGKEY KCKVSNKGLP  SSIEKTISKA   KGQPREPQVY TLPPSQEEMT KNQVSLTCLV    KGFYPSDIAV EWESNGQPEN NYKTTPPVLD    SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH   EALHNHYTQK SLSLSLGK

In some embodiments, the antibody or immunologically active fragmentthereof comprises a human IgG4 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 4.

In other embodiments, the human IgG4 Fc region is modified at amino acid235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In someembodiments, the human IgG4 Fc region is modified at amino acid Asn297(Kabat Numbering) to prevent to glycosylation of the antibody, e.g.,Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the humanIgG4 Fc region is lacks Lys447, which corresponds to residue 218 of SEQID NO: 4 (EU index of Kabat et al 1991 Sequences of Proteins ofImmunological Interest).

In some embodiments, the human IgG Fc region is modified to enhance FcRnbinding. Examples of Fc mutations that enhance binding to FcRn areMet252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively)(Kabat numbering, Dall'Acqua et al 2006, J. Biol Chem Vol 281(33)23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al2010 Nature Biotech, Vol 28(2) 157-159), Met252Ile, Thr256Asp, Met428Leu(M252I, T256D, M428L, respectively) or Met252Tyr, Met428Leu/Val (M252Y,M428L/V, respectively), (EU index of Kabat et al 1991 Sequences ofProteins of Immunological Interest). Met252 corresponds to residue 23 inSEQ ID NOs: 1, 3, and 4 and residue 22 in SEQ ID NO: 2. Ser254corresponds to corresponds to residue 25 in SEQ ID NOs: 1, 3, and 4 andresidue 24 in SEQ ID NO: 2. Thr256 corresponds to residue 27 in SEQ IDNOs: 1, 3, and 4 and residue 26 in SEQ ID NO: 2. Met428 corresponds toresidue 199 in SEQ ID NOs: 1, 3, and 4 and residue 198 in SEQ ID NO: 2.Asn434 corresponds to residue 205 in SEQ ID NOs: 1, 3, and 4 and residue204 in SEQ ID NO: 2.

In some embodiments, where the fusion protein of the invention includesan Fc polypeptide, the Fc polypeptide is mutated or modified. In theseembodiments, the mutated or modified Fc polypeptide includes thefollowing mutations: Met252Tyr and Met428Leu (M252Y, M428L) using theKabat numbering system.

In some embodiments, the human IgG Fc region is modified to alterantibody-dependent cellular cytotoxicity (ADCC) and/orcomplement-dependent cytotoxicity (CDC), e.g., the amino acidmodifications described in Natsume et al., 2008 Cancer Res, 68(10):3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al.,2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS, 103(11): 4005-4010,Shields et al., 2001 JBC, 276(9): 6591-6604; Stavenhagen et al., 2007Cancer Res, 67(18): 8882-8890; Stavenhagen et al., 2008 Advan. EnzymeRegul., 48: 152-164; Alegre et al, 1992 J Immunol, 148: 3461-3468;Reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11. Examples ofmutations that enhance ADCC include modification at Ser239 and Ile332,for example Ser239Asp and Ile332Glu (S239D, 1332E). Examples ofmutations that enhance CDC include modifications at Lys326 whichcorresponds to residue 97 of SEQ ID NOs: 1, 3, and 4 and residue 96 ofSEQ ID NO: 2, and Glu333, which corresponds to residue 104 of SEQ IDNOs: 1, 3, and 4 and residue 103 of SEQ ID NO: 2. In some embodiments,the Fc region is modified at one or both of these positions, for exampleLys326Ala and/or Glu333Ala (K326A and E333A) using the Kabat numberingsystem.

In some embodiments, the human IgG Fc region is modified to induceheterodimerization. For example, having an amino acid modificationwithin the CH3 domain at Thr366, which when replaced with a more bulkyamino acid, e.g., Try (T366W), is able to preferentially pair with asecond CH3 domain having amino acid modifications to less bulky aminoacids at positions Thr366, which corresponds to residue 137 of SEQ IDNOs: 1, 3, and 4 and residue 136 of SEQ ID NO: 2, Leu368, whichcorresponds to residue 139 of SEQ ID NOs: 1, 3, and 4 and residue 138 ofSEQ ID NO: 2, and Tyr407, which corresponds to residue 178 of SEQ IDNOs: 1, 3, and 4 and residue 177 of SEQ ID NO: 2, e.g., Ser, Ala andVal, respectively (T366S/L368A/Y407V). Heterodimerization via CH3modifications can be further stabilized by the introduction of adisulfide bond, for example by changing Ser354, which corresponds toresidue 125 of SEQ ID NOs: 1, 3, and 4 and residue 124 of SEQ ID NO: 2,to Cys (S354C) and Y349, which corresponds to residue 120 of SEQ ID NOs:1, 3, and 4 and residue 119 of SEQ ID NO: 2, to Cys (Y349C) on oppositeCH3 domains (Reviewed in Carter, 2001 Journal of Immunological Methods,248: 7-15). In some of these embodiments, the Fc region may be modifiedat the protein-A binding site on one member of the heterodimer so as toprevent protein-A binding and thereby enable more efficient purificationof the heterodimeric fusion protein. An exemplary modification withinthis binding site is Ile253, which corresponds to residue 24 of SEQ IDNOs: 1, 3, and 4 and residue 23 of SEQ ID NO: 2, for example Ile253Arg(I253R). For example the I253R modification maybe combined with eitherthe T366S/L368A/Y407V modifications or with the T366W modifications. TheT366S/L368A/Y407V modified Fc is capable of forming homodimers as thereis no steric occlusion of the dimerization interface as there is in thecase of the T336W modified Fc. Therefore, in preferred embodiments theI253R modification is combined with the T366S/L368A/Y407V modified Fc todisallow purification any homodimeric Fc that may have formed.

In some embodiments, the human IgG Fc region is modified to preventdimerization. In these embodiments, the fusion proteins of the presentinvention are monomeric. For example modification at residue Thr366 to acharged residue, e.g. Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu(T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3dimerization.

In some embodiments, the fusion protein contains a polypeptide derivedfrom an immunoglobulin hinge region. The hinge region can be selectedfrom any of the human IgG subclasses. For example the fusion protein maycontain a modified IgG1 hinge having the sequence of EPKSSDKTHTCPPC (SEQID NO: 5), where in the Cys220 that forms a disulfide with theC-terminal cysteine of the light chain is mutated to serine, e.g.,Cys220Ser (C220S). In other embodiments, the fusion protein contains atruncated hinge having a sequence DKTHTCPPC (SEQ ID NO: 6). In someembodiments, the fusion protein has a modified hinge from IgG4, which ismodified to prevent or reduce strand exchange, e.g., Ser228Pro (S228P),having the sequence ESKYGPPCPPC (SEQ ID NO: 7). In some embodiments, thefusion protein contains one or more linker polypeptides. In otherembodiments, the fusion protein contains one or more linker and one ormore hinge polypeptides.

In some embodiments, the fusion proteins of the present invention lackor have reduced Fucose attached to the N-linked glycan-chain at N297.There are numerous ways to prevent fucosylation, including but notlimited to production in a FUT8 deficient cell line; addition inhibitorsto the mammalian cell culture media, for example Castanospermine,2-deoxy-fucose, 2-flurofucose; the use of production cell lines withnaturally reduced fucosylation pathways, and metabolic engineering ofthe production cell line.

In some embodiments, the VPBP is engineered to eliminate recognition bypre-existing antibodies found in humans. In some embodiments, singledomain antibodies of the present invention are modified by mutation ofposition Leu11, for example Leu11Glu (L11E) or Leu11Lys (L11K). In otherembodiments, single domain antibodies of the present invention aremodified by changes in carboxy-terminal region, for example the terminalsequence consists of GQGTLVTVKPGG (SEQ ID NO: 8) or GQGTLVTVEPGG (SEQ IDNO: 9) or modification thereof. In some embodiments, the single domainantibodies of the present invention are modified by mutation of position11 and by changes in carboxy-terminal region.

In some embodiments, the VPBPs of the fusion proteins of the presentinvention are operably linked via amino acid linkers. In someembodiments, these linkers are composed predominately of the amino acidsGlycine and Serine, denoted as GS-linkers herein. The GS-linkers of thefusion proteins of the present invention can be of various lengths, forexample 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 aminoacids in length.

In some embodiments, the GS-linker comprises an amino acid sequenceselected from the group consisting of GGSGGS, i.e., (GGS)₂ (SEQ ID NO:75); GGSGGSGGS, i.e., (GGS)₃ (SEQ ID NO: 76); GGSGGSGGSGGS, i.e., (GGS)₄(SEQ ID NO: 77); GGSGGSGGSGGSGGS, i.e., (GGS)₅ (SEQ ID NO: 45), GGGGS(SEQ ID NO: 78); GGGGSGGGGS, i.e., (GGGGS2) (SEQ ID NO: 79), andGGGGSGGGGSGGGGS, i.e., (GGGGS3) (SEQ ID NO: 80).

In some embodiments, the fusion protein is tetravalent. In someembodiments, the tetravalent fusion protein has the following structure:VHH-Linker-VHH-Linker-Hinge-Fc, where the VHH is a humanized or fullyhuman VHH sequence. In some embodiments, the tetravalent fusion proteinhas the following structure: VHH-Linker-Hinge-Fc-Linker-VHH, where theVHH is a humanized or fully human VHH sequence.

In some embodiments, the fusion protein is hexavalent. In someembodiments, the hexavalent fusion protein has the following structure:VHH-Linker-VHH-Linker-VHH-Linker-Hinge-Fc, where the Willis a humanizedor fully human VHH sequence. In some embodiments, the hexavalent fusionprotein has the following structure:VHH-Linker-VHH-Linker-Hinge-Fc-Linker-VHH, orVHH-Linker-Hinge-Fc-Linker-VHH-Linker-VHH where the VHH is a humanizedor fully human VHH sequence.

In some embodiments, the fusion protein lacks an Fc region. In theseembodiments, wherein the fusion protein is tetravalent, the protein hasthe following structure VHH-Linker-VHH-Linker-VHH-Linker-VHH-Linker. Inthese embodiments, wherein the fusion protein is pentavalent, theprotein has the following structureVHH-Linker-VHH-Linker-VHH-Linker-VHH-Linker-VHH. In these embodiments,wherein the fusion protein is hexavalent, the protein has the followingstructure VHH-Linker-VHH-Linker-VHH-Linker-VHH-Linker-VHH-Linker-VHH. Inthese embodiments, the VHH is a humanized or fully human VHH sequence.

In some embodiments, the VPBP-containing fusion protein may also containadditional binding domains that recognize non-V-tip proteins of gramnegative bacteria such as PcrV from Pseudomonas aeruginosa. Theseadditional bacterial binding domains may confer additional functionalityto the fusion protein of the present invention. These additionalfunctionalities may include neutralization of additional bacterialvirulence or growth factors or enable opsono-phagocytosis of thebacteria by host phagocytic cells. In some embodiments, theVPBP-containing fusion protein may also contain additional bindingdomains that recognize non-bacterial proteins. These additionalnon-bacterial binding domains, may confer additional functionality tothe fusion protein of the present invention. These additionalfunctionalities may enhance immune cell recruitment or activation,including neutrophils, natural killer cells, macrophages, monocytes,dendritic cells and T-cells.

In some embodiments, the VPBP-containing fusion protein may also containadditional binding domains that recognize the outer membrane protein I(OprI) protein or a fragment thereof. In a preferred embodiment, theVPBP-containing fusion protein includes at least a first domain thatbinds PcrV or a fragment thereof and a second domain that binds OprI ora fragment thereof. These bispecific fusion proteins are referred toherein as “PcrV×OprI bispecific fusion proteins,” “PcrV×OprI fusionproteins” and/or “PcrV×OprI fusions.” OprI is a cell surface proteinthat is highly conserved amongst P. aeruginosa strains. OprI is anchoredto outer membrane via N-term Cys-lipidation, is present in 100% of P.aeruginosa strains tested, and is 100% conserved in genome sequenced P.aeruginosa strains.

In some embodiments, the first domain comprises one or more sequencesfrom the PcrV sequences shown in Table 1. In some embodiments, thesecond domain comprises one or more sequences that bind OprI. In someembodiments, the second domain comprises one or more sequences from theOprI sequences shown in Table 2.

Dual targeting of PcrV and OprI allows the fusion polypeptides to tetheror otherwise attach and/or bind to the bacteria cell surface, and itprovides enhanced protection in vivo.

In some embodiments, the V-tip protein targeting molecule comprises oneor more sequences from the PcrV sequences shown in Table 1.

The molecules provided herein exhibit inhibitory activity, for exampleby inhibiting at least one biological activity of one or more V-tipproteins of Gram negative bacteria, such as for example, functionaltranslocalization of the effector molecules across target cellmembranes. The molecules provided herein completely or partially reduceor otherwise modulate expression or activity of one or more V-tipproteins of Gram negative bacteria upon binding to, or otherwiseinteracting with, the V-tip protein(s) such as PcrV from Pseudomonasaeruginosa. The reduction or modulation of a biological function of oneor more V-tip proteins of Gram negative bacteria is complete or partialupon interaction between the molecules and the V-tip protein(s). Themolecules are considered to completely inhibit expression or activity ofone or more V-tip proteins of Gram negative bacteria when the level ofexpression or activity of the V-tip protein(s) in the presence of themolecule is decreased by at least 95%, e.g., by 96%, 97%, 98%, 99% or100% as compared to the level of expression or activity of the V-tipprotein(s) in the absence of interaction, e.g., binding, with a moleculedescribed herein. The molecules are considered to partially inhibitexpression or activity one or more V-tip proteins of Gram negativebacteria when the level of expression or activity of the V-tipprotein(s) in the presence of the molecule is decreased by less than95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% ascompared to the level of expression or activity of the V-tip protein(s)in the absence of interaction, e.g., binding, with a molecule describedherein.

The V-tip protein targeting molecules provided herein are useful intreating, alleviating a symptom of, ameliorating and/or delaying theprogression of a disease or disorder in a subject suffering from oridentified as being at risk for a disease or disorder associated with atleast one biological activity of one or more V-tip proteins of Gramnegative bacteria such as PcrV from Pseudomonas aeruginosa, such as, forexample, functional translocalization of the effector molecules acrosstarget cell membranes.

The disclosure provides molecules, e.g., polypeptides includingantibodies, antigen-binding antibody fragments, antibody-likepolypeptides, and/or fusion polypeptides, and compositions that bindbacterial non-V-tip proteins of the type III secretion system of Gramnegative bacteria, such as OprI from Pseudomonas aeruginosa, and methodsof making and using these compositions in a variety of therapeutic,diagnostic, and/or prophylactic indications. The OprI-targetingmolecules of the present invention are monospecific or multispecific andcan bind their target antigen(s) in a monovalent or multivalent manner.

In some embodiments, the OprI-protein targeting molecules are antibodiesand antibody-like molecules that specifically bind OprI. In someembodiments, the antibody or antigen-binding fragment thereof arederived from antibodies or antigen-binding antibody fragments including,for example, single-chain variable fragments (scFv), Fab fragments,single domain antibodies (sdAb), V_(NAR), or VHHs. In some embodiments,the anti-OprI antibodies are human or humanized sdAb. The sdAb fragmentscan be derived from VHH, V_(NAR), engineered VH or VK domains. VHHs canbe generated from camelid heavy chain only antibodies. VNARS can begenerated from cartilaginous fish heavy chain only antibodies. Variousmethods have been implemented to generate monomeric sdAbs fromconventionally heterodimeric VH and VK domains, including interfaceengineering and selection of specific germline families.

In other embodiments, the anti-OprI targeting molecules are derived fromnon-antibody scaffold proteins for example but not limited to designedankyrin repeat proteins (darpins), avimer, anticalin/lipocalins,centyrins and fynomers.

In some embodiments, the anti-OprI targeting molecule is an antibody orantigen-binding fragment thereof comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 46-70 and 88. In someembodiments, the anti-OprI targeting antibody or antigen-bindingfragment thereof also comprises an immunoglobulin Fc region orimmunologically active fragment thereof. In some embodiments, theanti-OprI targeting antibody or antigen-binding fragment thereof alsocomprises an immunoglobulin Fc region or immunologically active fragmentthereof comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-4.

In some embodiments, the fusion protein of the present invention iscomposed of a single polypeptide. In other embodiments, the fusionprotein of the present invention is composed of more than onepolypeptide. For example, wherein a heterodimerization domain isincorporated into the fusion protein so as the construct an asymmetricfusion protein. For example if an immunoglobulin Fc region isincorporated into the fusion protein the CH3 domain can be used ashomodimerization domain, or the CH3 dimer interface region can bemutated so as to enable heterodimerization.

In some embodiments, the fusion protein contains the VPBPs on oppositeends. For example the VPBPs are located on both the amino-terminal(N-terminal) portion of the fusion protein and the carboxy-terminal(C-terminal) portion of the fusion protein. In other embodiments, allthe VPBPs reside on the same end of the fusion protein. For example,VPBPs reside on either the amino or carboxyl terminal portions of thefusion protein.

In some embodiments, the present invention includes fusion proteinsincorporating more than one OprI targeting sequence and are referred toherein as multivalent. In some embodiments, the OprI targeting sequencesof the fusion protein recognize the same epitope on OprI and arereferred to herein as monospecific-multivalent. In other embodiments,the OprI targeting sequences of the fusion protein recognize distinctepitopes on OprI and are referred to herein asmultispecific-multivalent. In some embodiments, the OprI targetingsequence-containing fusion protein includes two OprI targeting sequencesand has a bivalent binding capacity toward the OprI. In someembodiments, the OprI targeting sequence-containing fusion proteinincludes three OprI targeting sequences and has a trivalent bindingcapacity toward the OprI. In some embodiments, the OprI targetingsequence-containing fusion protein includes four OprI targetingsequences and has a tetravalent binding capacity toward the OprI. Insome embodiments, the OprI targeting sequence-containing fusion proteinincludes six OprI targeting sequences and has a hexavalent bindingcapacity toward the OprI. In some embodiments, the OprI targetingsequence-containing fusion protein includes eight OprI targetingsequences and has an octavalent binding capacity toward the OprI. Inthese embodiments, the OprI targeting sequences incorporated into thefusion protein of the present invention can be monospecific ormultispecific.

In some embodiments, the fusion protein lacks an Fc region.

In some embodiments, the fusion protein comprises an immunoglobulin Fcregion or immunologically active fragment thereof comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 1-4.

In some embodiments, the Fc region of the OprI targeting antibody orantigen-binding fragment thereof or the OprI-targeting fusionpolypeptide includes a human IgG1 region. In some embodiments, humanIgG1 Fc region is modified at amino acid Asn297 (Boxed, Kabat Numbering)to prevent glycosylation of the antibody and/or fusion protein, e.g.,Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the Fcregion of the antibody and/or fusion protein is modified at amino acidLeu235 (Boxed, Kabat Numbering) to alter Fc receptor interactions, e.g.,Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the Fcregion of the antibody and/or fusion protein is modified at amino acidLeu234 (Boxed, Kabat Numbering) to alter Fc receptor interactions, e.g.,Leu234Ala (L234A). In some embodiments, the Fc region of the antibodyand/or fusion protein is altered at both amino acid 234 and 235, e.g.,Leu234Ala and Leu235Ala (L234A/L235A) or Leu234Val and Leu235Ala(L234V/L235A). In some embodiments, the Fc region of the antibody and/orfusion protein is lacking an amino acid at one or more of the followingpositions to reduce Fc receptor binding: Glu233 (E233, Bold in SEQ IDNO: 1), Leu234 (L234), or Leu235 (L235). In some embodiments, the Fcregion of the antibody and/or fusion protein is altered at Gly235 toreduce Fc receptor binding. For example, wherein Gly235 is deleted fromthe antibody and/or fusion protein. In some embodiments, the human IgG1Fc region is modified at amino acid Gly236 to enhance the interactionwith CD32A, e.g., Gly236Ala (G236A, Boxed in SEQ ID NO: 1). In someembodiments, the human IgG1 Fc region is lacks Lys447, which correspondsto residue 218 of SEQ ID NO: 1 (EU index of Kabat et al 1991 Sequencesof Proteins of Immunological Interest).

In some embodiments, the Fc region of the OprI targeting antibody orantigen-binding fragment thereof or the OprI-targeting fusionpolypeptide includes a human IgG2 region. In some embodiments, the humanIgG2 Fc region is modified at amino acid Asn297 (Boxed, to prevent toglycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp(N297D). In some embodiments, the human IgG2 Fc region lacks Lys447,which corresponds to residue 217 of SEQ ID NO: 2 (EU index of Kabat etal 1991 Sequences of Proteins of Immunological Interest).

In some embodiments, the Fc region of the OprI targeting antibody orantigen-binding fragment thereof or the OprI-targeting fusionpolypeptide includes a human IgG3 region. In some embodiments, the humanIgG3 Fc region is modified at amino acid Asn297 (Boxed, Kabat Numbering)to prevent to glycosylation of the antibody, e.g., Asn297Ala (N297A) orAsn297Asp (N297D). In some embodiments, the human IgG3 Fc region ismodified at amino acid 435 to extend the half-life, e.g., Arg435His(R435H, boxed in SEQ ID NO: 3). In some embodiments, the human IgG3 Fcregion is lacks Lys447, which corresponds to residue 218 of SEQ ID NO: 3(EU index of Kabat et al 1991 Sequences of Proteins of ImmunologicalInterest).

In some embodiments, the Fc region of the OprI targeting antibody orantigen-binding fragment thereof or the OprI-targeting fusionpolypeptide includes a human IgG4 region. In other embodiments, thehuman IgG4 Fc region is modified at amino acid 235 to alter Fc receptorinteractions, e.g., Leu235Glu (L235E). In some embodiments, the humanIgG4 Fc region is modified at amino acid Asn297 (Kabat Numbering) toprevent to glycosylation of the antibody, e.g., Asn297Ala (N297A) orAsn297Asp (N297D). In some embodiments, the human IgG4 Fc region islacks Lys447, which corresponds to residue 218 of SEQ ID NO: 4 (EU indexof Kabat et al 1991 Sequences of Proteins of Immunological Interest).

In some embodiments, the human IgG Fc region of the OprI targetingantibody or antigen-binding fragment thereof or the OprI-targetingfusion polypeptide is modified to enhance FcRn binding. Examples of Fcmutations that enhance binding to FcRn are Met252Tyr, Ser254Thr,Thr256Glu (M252Y, S254T, T256E, respectively) (Kabat numbering,Dall'Acqua et al 2006, J. Biol Chem Vol 281(33) 23514-23524), Met428Leuand Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol28(2) 157-159), Met252Ile, Thr256Asp, Met428Leu (M252I, T256D, M428L,respectively) or Met252Tyr, Met428Leu/Val (M252Y, M428L/V,respectively), (EU index of Kabat et al 1991 Sequences of Proteins ofImmunological Interest). Met252 corresponds to residue 23 in SEQ ID NOs:1, 3, and 4 and residue 22 in SEQ ID NO: 2. Ser254 corresponds tocorresponds to residue 25 in SEQ ID NOs: 1, 3, and 4 and residue 24 inSEQ ID NO: 2. Thr256 corresponds to residue 27 in SEQ ID NOs: 1, 3, and4 and residue 26 in SEQ ID NO: 2. Met428 corresponds to residue 199 inSEQ ID NOs: 1, 3, and 4 and residue 198 in SEQ ID NO: 2. Asn434corresponds to residue 205 in SEQ ID NOs: 1, 3, and 4 and residue 204 inSEQ ID NO: 2.

In some embodiments, the Fc region of the OprI targeting antibody orantigen-binding fragment thereof or the OprI-targeting fusionpolypeptide is mutated or modified. In these embodiments, the mutated ormodified Fc polypeptide includes the following mutations: Met252Tyr andMet428Leu (M252Y, M428L) using the Kabat numbering system.

In some embodiments, the human IgG Fc region of the OprI targetingantibody or antigen-binding fragment thereof or the OprI-targetingfusion polypeptide is modified to alter antibody-dependent cellularcytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC),e.g., the amino acid modifications described in Natsume et al., 2008Cancer Res, 68(10): 3863-72; Idusogie et al., 2001 J Immunol, 166(4):2571-5; Moore et al., 2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS,103(11): 4005-4010, Shields et al., 2001 JBC, 276(9): 6591-6604;Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Stavenhagen etal., 2008 Advan. Enzyme Regul., 48: 152-164; Alegre et al, 1992 JImmunol, 148: 3461-3468; Reviewed in Kaneko and Niwa, 2011 Biodrugs,25(1):1-11. Examples of mutations that enhance ADCC include modificationat Ser239 and Ile332, for example Ser239Asp and Ile332Glu (S239D,1332E). Examples of mutations that enhance CDC include modifications atLys326 which corresponds to residue 97 of SEQ ID NOs: 1, 3, and 4 andresidue 96 of SEQ ID NO: 2, and Glu333, which corresponds to residue 104of SEQ ID NOs: 1, 3, and 4 and residue 103 of SEQ ID NO: 2. In someembodiments, the Fc region is modified at one or both of thesepositions, for example Lys326Ala and/or Glu333Ala (K326A and E333A)using the Kabat numbering system.

In some embodiments, the human IgG Fc region of the OprI targetingantibody or antigen-binding fragment thereof or the OprI-targetingfusion polypeptide is modified to induce heterodimerization. Forexample, having an amino acid modification within the CH3 domain atThr366, which when replaced with a more bulky amino acid, e.g., Try(T366W), is able to preferentially pair with a second CH3 domain havingamino acid modifications to less bulky amino acids at positions Thr366,which corresponds to residue 137 of SEQ ID NOs: 1, 3, and 4 and residue136 of SEQ ID NO: 2, Leu368, which corresponds to residue 139 of SEQ IDNOs: 1, 3, and 4 and residue 138 of SEQ ID NO: 2, and Tyr407, whichcorresponds to residue 178 of SEQ ID NOs: 1, 3, and 4 and residue 177 ofSEQ ID NO: 2, e.g., Ser, Ala and Val, respectively (T366S/L368A/Y407V).Heterodimerization via CH3 modifications can be further stabilized bythe introduction of a disulfide bond, for example by changing Ser354,which corresponds to residue 125 of SEQ ID NOs: 1, 3, and 4 and residue124 of SEQ ID NO: 2, to Cys (S354C) and Y349, which corresponds toresidue 120 of SEQ ID NOs: 1, 3, and 4 and residue 119 of SEQ ID NO: 2,to Cys (Y349C) on opposite CH3 domains (Reviewed in Carter, 2001 Journalof Immunological Methods, 248: 7-15). In some of these embodiments, theFc region may be modified at the protein-A binding site on one member ofthe heterodimer so as to prevent protein-A binding and thereby enablemore efficient purification of the heterodimeric fusion protein. Anexemplary modification within this binding site is Ile253, whichcorresponds to residue 24 of SEQ ID NOs: 1, 3, and 4 and residue 23 ofSEQ ID NO: 2, for example Ile253Arg (I253R). For example the I253Rmodification maybe combined with either the T366S/L368A/Y407Vmodifications or with the T366W modifications. The T366S/L368A/Y407Vmodified Fc is capable of forming homodimers as there is no stericocclusion of the dimerization interface as there is in the case of theT336W modified Fc. Therefore, in preferred embodiments the I253Rmodification is combined with the T366S/L368A/Y407V modified Fc todisallow purification any homodimeric Fc that may have formed.

In some embodiments, the human IgG Fc region of the OprI targetingantibody or antigen-binding fragment thereof or the OprI-targetingfusion polypeptide is modified to prevent dimerization. In theseembodiments, the antibodies and/or fusion proteins of the presentinvention are monomeric. For example modification at residue Thr366 to acharged residue, e.g. Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu(T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3dimerization.

In some embodiments, the fusion proteins of the present invention areoperably linked via amino acid linkers. In some embodiments, theselinkers are composed predominately of the amino acids Glycine andSerine, denoted as GS-linkers herein. The GS-linkers of the fusionproteins of the present invention can be of various lengths, for example5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids inlength.

In some embodiments, the GS-linker comprises an amino acid sequenceselected from the group consisting of GGSGGS, i.e., (GGS)₂ (SEQ ID NO:75); GGSGGSGGS, i.e., (GGS)₃ (SEQ ID NO: 76); GGSGGSGGSGGS, i.e., (GGS)₄(SEQ ID NO: 77); GGSGGSGGSGGSGGS, i.e., (GGS)₅ (SEQ ID NO: 45), GGGGS(SEQ ID NO: 78); GGGGSGGGGS, i.e., (GGGGS2) (SEQ ID NO: 79), andGGGGSGGGGSGGGGS, i.e., (GGGGS3) (SEQ ID NO: 80).

In some embodiments, the fusion protein is tetravalent. In someembodiments, the tetravalent fusion protein has the following structure:VHH-Linker-VHH-Linker-Hinge-Fc, where the VHH is a humanized or fullyhuman VHH sequence. In some embodiments, the tetravalent fusion proteinhas the following structure: VHH-Linker-Hinge-Fc-Linker-VHH, where theVHH is a humanized or fully human VHH sequence.

In some embodiments, the fusion protein is hexavalent. In someembodiments, the hexavalent fusion protein has the following structure:VHH-Linker-VHH-Linker-VHH-Linker-Hinge-Fe, where the VHH is a humanizedor fully human VHH sequence. In some embodiments, the hexavalent fusionprotein has the following structure:VHH-Linker-VHH-Linker-Hinge-Fc-Linker-VHH, orVHH-Linker-Hinge-Fc-Linker-VHH-Linker-VHH where the VHH is a humanizedor fully human VHH sequence.

In some embodiments, the fusion protein lacks an Fc region. In theseembodiments, wherein the fusion protein is tetravalent, the protein hasthe following structure VHH-Linker-VHH-Linker-VHH-Linker-VHH-Linker. Inthese embodiments, wherein the fusion protein is pentavalent, theprotein has the following structureVHH-Linker-VHH-Linker-VHH-Linker-VHH-Linker-VHH. In these embodiments,wherein the fusion protein is hexavalent, the protein has the followingstructure VHH-Linker-VHH-Linker-VHH-Linker-VHH-Linker-VHH-Linker-VHH. Inthese embodiments, the VHH is a humanized or fully human VHH sequence.

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablecarriers, buffers, excipients, and other agents that are incorporatedinto formulations to provide improved transfer, delivery, tolerance, andthe like. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman WN “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a series of schematic representations of exemplaryVPBP-containing fusion proteins of the present disclosure. VPBPrecognizing distinct epitopes are differentially shaded in theseschematic representations.

FIGS. 2A, 2B, and 2C are a series of graphs depicting hemolysis analysisusing various VPBP-containing fusion proteins of the disclosure.

FIGS. 3A and 3B are a series of graphs depicting cytotoxicity analysisusing various VPBP-containing fusion proteins of the disclosure. TheA549 cell line was used as the target cell line.

FIGS. 4A, 4B, and 4C are a series of graphs depicting survival analysisin an infection model using various VPBP-containing fusion proteins ofthe disclosure. The V2L2 mAb was used a positive control as PCRVblocking antibody.

FIG. 5 is a graph depicting binding of an example OprI antibody to bindto a variety of Pseudomonas aeruginosa strains and to Pseudomonasputida.

FIG. 6 is a graph depicting the ability of various VPBP-containingfusion proteins of the disclosure to bind P. aeruginosa via OprI.

FIG. 7 is a graph depicting the ability of various VPBP-containingfusion proteins of the disclosure to provide superior protection in vivoin P. aeruginosa prophylaxis-pneumonia model. Included herein is anexemplary multispecific, PCRV-OprI (PCRV-18-15-OprI-7), fusion proteinof the disclosure demonstrating enhanced protective capacity over abispecific targeting PCRV and PSL (disclosed in US20150284450 andDiGiandomenico et al., “A multifunctional bispecific antibody protectsagainst Pseudomonas aeruginosa,” Sci Transl Med., vol. 6(262): 262ra155(2014)) at equivalent molar dose.

DETAILED DESCRIPTION

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients. The term patientincludes human and veterinary subjects.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the terms “targeting fusion protein” and “antibody” canbe synonyms. As used herein, the term “antibody” refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin (Ig) molecules, i.e., molecules that contain an antigenbinding site that specifically binds (immunoreacts with) an antigen. By“specifically bind” or “immunoreacts with” “or directed against” ismeant that the antibody reacts with one or more antigenic determinantsof the desired antigen and does not react with other polypeptides orbinds at much lower affinity (K_(d)>10′). Antibodies include, but arenot limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody),single chain, Fab, Fab′ and F(ab′)2 fragments, F_(v), scFvs, an Fabexpression library, and single domain antibody (sdAb) fragments, forexample V_(H)H, V_(NAR), engineered V_(H) or V_(K).

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses (also known as isotypes) as well, such as IgG₁, IgG₂, andothers. Furthermore, in humans, the light chain may be a kappa chain ora lambda chain.

The term “monoclonal antibody” (mAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three-dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

The single domain antibody (sdAb) fragments portions of the fusionproteins of the present invention are referred to interchangeably hereinas targeting polypeptides herein.

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to/by an immunoglobulin or fragment thereof,or a T-cell receptor. The term “epitope” includes any proteindeterminant capable of specific binding to/by an immunoglobulin orT-cell receptor. Epitopic determinants usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics. An antibodyis said to specifically bind an antigen when the dissociation constantis ≤1 μM; e.g., ≤100 nM, preferably ≤10 nM and more preferably ≤1 nM.

As used herein, the terms “immunological binding” and “immunologicalbinding properties” and “specific binding” refer to the non-covalentinteractions of the type which occur between an immunoglobulin moleculeand an antigen for which the immunoglobulin is specific. The strength,or affinity of immunological binding interactions can be expressed interms of the dissociation constant (K_(d)) of the interaction, wherein asmaller K_(d) represents a greater affinity. Immunological bindingproperties of selected polypeptides can be quantified using methods wellknown in the art. One such method entails measuring the rates ofantigen-binding site/antigen complex formation and dissociation, whereinthose rates depend on the concentrations of the complex partners, theaffinity of the interaction, and geometric parameters that equallyinfluence the rate in both directions. Thus, both the “on rate constant”(k_(on)) and the “off rate constant” (k_(off)) can be determined bycalculation of the concentrations and the actual rates of associationand dissociation. (See Nature 361:186-87 (1993)). The ratio ofk_(off)/k_(on) enables the cancellation of all parameters not related toaffinity, and is equal to the dissociation constant K_(d). (See,generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). Anantibody of the present invention is said to specifically bind to anantigen, when the equilibrium binding constant (K_(d)) is ≤1 μM,preferably ≤100 nM, more preferably ≤10 nM, and most preferably ≤100 pMto about 1 pM, as measured by assays such as radioligand binding assays,surface plasmon resonance (SPR), flow cytometry binding assay, orsimilar assays known to those skilled in the art.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine valine,glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic amino acids are aspartate, glutamate; (2)basic amino acids are lysine, arginine, histidine; (3) non-polar aminoacids are alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, and (4) uncharged polar amino acids are glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Thehydrophilic amino acids include arginine, asparagine, aspartate,glutamine, glutamate, histidine, lysine, serine, and threonine. Thehydrophobic amino acids include alanine, cysteine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan, tyrosine and valine.Other families of amino acids include (i) serine and threonine, whichare the aliphatic-hydroxy family; (ii) asparagine and glutamine, whichare the amide containing family; (iii) alanine, valine, leucine andisoleucine, which are the aliphatic family; and (iv) phenylalanine,tryptophan, and tyrosine, which are the aromatic family. For example, itis reasonable to expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting molecule, especially if the replacement doesnot involve an amino acid within a framework site. Whether an amino acidchange results in a functional peptide can readily be determined byassaying the specific activity of the polypeptide derivative. Assays aredescribed in detail herein. Fragments or analogs of antibodies orimmunoglobulin molecules can be readily prepared by those of ordinaryskill in the art. Preferred amino- and carboxy-termini of fragments oranalogs occur near boundaries of functional domains. Structural andfunctional domains can be identified by comparison of the nucleotideand/or amino acid sequence data to public or proprietary sequencedatabases. Preferably, computerized comparison methods are used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure and/or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al. Science 253:164 (1991). Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in accordance with theinvention.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long’ morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long. The term“analog” as used herein refers to polypeptides which are comprised of asegment of at least 25 amino acids that has substantial identity to aportion of a deduced amino acid sequence and which has specific bindingto CD47, under suitable binding conditions. Typically, polypeptideanalogs comprise a conservative amino acid substitution (or addition ordeletion) with respect to the naturally-occurring sequence. Analogstypically are at least 20 amino acids long, preferably at least 50 aminoacids long or longer, and can often be as long as a full-lengthnaturally-occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986), Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987). Such compounds are often developed with theaid of computerized molecular modeling. Peptide mimetics that arestructurally similar to therapeutically useful peptides may be used toproduce an equivalent therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity), such as human antibody, but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of:—CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis and trans), —COCH₂—,CH(OH)CH₂—, and —CH₂SO—, by methods well known in the art. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g., D-lysine in place of L-lysine) maybe used to generate more stable peptides. In addition, constrainedpeptides comprising a consensus sequence or a substantially identicalconsensus sequence variation may be generated by methods known in theart (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example,by adding internal cysteine residues capable of forming intramoleculardisulfide bridges which cyclize the peptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, and/or an extractmade from biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing and/or ameliorating a disorder and/or symptomsassociated therewith. By “alleviate” and/or “alleviating” is meantdecrease, suppress, attenuate, diminish, arrest, and/or stabilize thedevelopment or progression of a disease such as, for example, a cancer.It will be appreciated that, although not precluded, treating a disorderor condition does not require that the disorder, condition or symptomsassociated therewith be completely eliminated.

In this disclosure, “comprises,” “comprising,” “containing,” “having,”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; the terms“consisting essentially of” or “consists essentially” likewise have themeaning ascribed in U.S. Patent law and these terms are open-ended,allowing for the presence of more than that which is recited so long asbasic or novel characteristics of that which is recited are not changedby the presence of more than that which is recited, but excludes priorart embodiments.

By “effective amount” is meant the amount required to ameliorate thesymptoms of a disease relative to an untreated patient. The effectiveamount of active compound(s) used to practice the present invention fortherapeutic treatment of a disease varies depending upon the manner ofadministration, the age, body weight, and general health of the subject.Ultimately, the attending physician or veterinarian will decide theappropriate amount and dosage regimen. Such amount is referred to as an“effective” amount.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, rodent, ovine,primate, camelid, or feline.

The term “administering,” as used herein, refers to any mode oftransferring, delivering, introducing, or transporting a therapeuticagent to a subject in need of treatment with such an agent. Such modesinclude, but are not limited to, oral, topical, intravenous,intraperitoneal, intramuscular, intradermal, intranasal, andsubcutaneous administration.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Unless specifically stated or obvious from context, as used herein, theterms “a,” “an,” and “the” are understood to be singular or plural.Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromthe context, all numerical values provided herein are modified by theterm “about.”

Therapeutic formulations of the invention, which include a V-tip proteintargeting molecule of the invention, are used to treat or alleviate asymptom associated with a disease or disorder associated with aberrantactivity and/or expression of one or more V-tip proteins of Gramnegative bacteria, such as PcrV from Pseudomonas aeruginosa, in asubject. A therapeutic regimen is carried out by identifying a subject,e.g., a human patient suffering from (or at risk of developing) adisease or disorder associated with aberrant activity and/or expressionof one or more V-tip proteins of Gram negative bacteria, such as PcrVfrom Pseudomonas aeruginosa, using standard methods, including any of avariety of clinical and/or laboratory procedures. The term patientincludes human and veterinary subjects. The term subject includes humansand other mammals.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular disease or disorderassociated with aberrant activity and/or expression of one or more V-tipproteins of Gram negative bacteria, such as PcrV from Pseudomonasaeruginosa. Alleviation of one or more symptoms of the disease ordisorder associated with aberrant activity and/or expression of one ormore V-tip proteins of Gram negative bacteria, such as PcrV fromPseudomonas aeruginosa, indicates that the V-tip protein targetingmolecule confers a clinical benefit.

Methods for the screening of V-tip protein targeting molecules thatpossess the desired specificity include, but are not limited to, enzymelinked immunosorbent assay (ELISA), enzymatic assays, flow cytometry,and other immunologically mediated techniques known within the art.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

Example 1: Hemolysis Blocking

The ability of the VPBPs of the present invention to block bacterialinduced hemolysis of red blood cells (RBCs) can be assessed by numerousprotocols known in the art. For example, human RBCs were washed in PBSand resuspended at 2% (v/v) in DMEM. Pseudomonas aeruginosa bacteriaplus serially diluted antibodies were added to RBCs in 96 well roundbottom plates. The plates were incubated for 2 h at 37° C. and then 2 hat 4° C. The plates were then spun to pellet intact RBCs, after whichthe supernatant was transferred to a flat bottom 96-well plate forspectrophotometric observation of released hemoglobin.

As shown in FIGS. 2A-2D, both monospecific and multispecific multivalentVPBPs of the present invention are able to block bacterial inducedhemolysis of RBCs.

Example 2: Cytotoxicity Blocking

The ability of the VPBPs of the present invention to block bacterialinduced cytotoxicity of mammalian cells can be assessed by numerousprotocols known in the art. For example, A confluent monolayer of A549(lung epithelial) cells were grown in 96-well plates. Cells were loadedwith Calcein AM and then washed to remove excess Calcein. P. aeruginosaand antibodies at varying concentrations were added to the A549 cellsand incubated for 2 h at 37° C. Monolayers were then washed, after whichthe remaining cells were quantified by fluorescence.

As shown in FIGS. 3A-3B, both monospecific and multispecific multivalentVPBPs of the present invention are able to block bacterial inducedcytotoxicity of mammalian cells.

Example 3: Pseudomonas aeruginosa Infection Model

The ability of the VPBPs of the present invention to protect against abacterial infection can be assessed using a mouse model of P. aeruginosainfection. Mice were pre-treated with PcrV antibodies 24 h prior toinfection with P. aeruginosa. At t=0 mice were intra-tracheally infectedwith P. aeruginosa and survival was monitored for 4 days. Importantly,it was discovered that the multispecific multivalent VPBP-containingfusion proteins conferred substantially more protection compared tomonospecific multivalent VPBP-containing fusion proteins (FIGS. 4A-4C).The multispecific multivalent VPBP-containing fusions of the presentinvention also are substantially more potent than the anti-PCRVantibody, V2L2, known in the art to be a potent blocker of P. aeruginosainduced hemolysis (see e.g., PCT/US2012/063639, published as WO2013/0170565).

Example 4: OprI Antibodies Bind to Multiple Strains

The ability of OprI targeting antibodies to bind to Pseudomonas strainscan be assessed by whole cell bacterial ELISA. Bacterial cultures weregrown to mid-logarithmic phase in standard bacteriologic media, thenwashed and resuspended in PBS. Equal volumes of bacterial suspensionwere placed in 96 well plates and incubated at 37C for 24 h. Plates wereblocked with BSA and then serial dilutions of antibodies were added.After subsequent washing, HRP-conjugated anti-human Fc specificsecondary antibody was added. Following incubation and washing, TMBsubstrate was added and absorbance at 600 nm was measured to detectbinding of antibodies to bacteria. As shown in FIG. 5, OprI antibodieswere found to bind to all strains of Pseudomonas aeruginosa tested, aswell as Pseudomonas putida.

Example 5: Bispecific Molecules in Pseudomonas aeruginosa InfectionModel

The studies presented herein demonstrate the ability of the VPBPs of thepresent invention to protect against a bacterial infection can beassessed using a mouse model of P. aeruginosa infection. In particular,these studies use a VPBP that binds both PcrV and outer membrane proteinI (“OprI”), also referred to herein as “PcrV×OprI bispecific fusionproteins,” “PcrV×OprI fusion proteins” and/or “PcrV×OprI fusions.” Dualtargeting of PcrV and OprI allows the fusion polypeptides to tether orotherwise attach and/or bind to the bacteria cell surface, and thestudies provided herein demonstrate that this dual-targeting alsoproduces enhanced protection in vivo.

The PcrV×OprI bispecific fusions target the bacterial cell surface bytargeting OprI. FIG. 6 demonstrates that the PcrV×OprI bispecificfusions of the disclosure bind to P. aeruginosa by flow cytometry.

The PcrV×OprI bispecific fusions also are more potent in vivo than thebispecific molecule Bis4, which binds PcrV and PSL and is known in theart to be a blocker P. aeruginosa induced hemolysis (see e.g.,DiGiandomenico et al., “A multifunctional bispecific antibody protectsagainst Pseudomonas aeruginosa,” Sci Transl Med., vol. 6(262): 262ra155(2014)). FIG. 7 demonstrates that the PcrV×OprI bispecific fusionproteins provide superior protection in vivo in a P. aeruginosapneumonia-prophylaxis animal model.

TABLE 1 PcrV-VPBP Sequences PcrV1A (1A7)EVQLVQSGGGLVQAGGSLRLSCAASGRIFGTYGMGWFRQAPGKERVFVAAISKSGPTTYYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCGASSHSMLVVTTSQVDYWGRGTQVTVSS (SEQ ID NO: 10) PcrV2A (1B9)EVQLVQSGGGLVQPGGSLRLSCAVSGLIFDNYGIGWFRQAPEKEREGVSCIHESDGSTYYTDSVKGRFAISRDNAKNTGYLEMNNLKPEDTAVYYCVVLSYVSRCPEGSKYDYWGQGTQVTVSS (SEQ ID NO: 11) PcrV3A (1B12)EVQLVQSGGGLVQPGGSLRLSCAASGFTLDYYPIGWFRQAPGKEREGVSCISSSEGSTYYADSVKGRFTISRDNAKNTVYLQMNNMKPEDTAVYYCATDFFTTGCPSGGGKYDYWGQGTQVTVSS (SEQ ID NO: 12) PcrV4A (1G9)EVQLVQSGGGLVRAGGSLRLSCAPSERTFGSFGMGWFRQAPGKEREFVAALMWGTSYTSYADSVKGRFTVSKDNAKNTLYLQMNSLKPEDTAVYYCAAGAVGADPRRYDYWGQGTQVTVSS (SEQ ID NO: 13) PcrV5A (2A5)EVQLVQSGGGLVQAGGSLRLSCAASGLAFRNYRMGWFRQAPGKEREFVAAISGNIGGSGVGTDYADSVKGRFTISRDNDKDTAYLQMNSLKPEDTAVYYCAADHEILTMLPGEYDFWGEGTQVTVSS (SEQ ID NO: 14) PcrV6A (2A11)EVQLVQSGGGLVQPGGSLRLSCAASGSTLDYYAIGWFRQAPGKEREGVACISSSDGSTDYADSMKGRFTISRDNAQKTVYLQMNSLKPEDTAVYSCAAVAFFCGSSWYLSSGMDYWGKGTQVTVSS (SEQ ID NO: 15) PcrV7A (2A12)EVQLVQSGGGLVQAGGSLRLSCAASGGTFSSNAMYWYRQAPGKQRELVASISGTSNANYPDSVKGRFTISRDNAKNTVTLQMNSLKPEDTAVYYCRAAPVSGPLIGRIFWGQGTQVTVSS (SEQ ID NO: 16) PcrV8A (2B4)EVQLVQSGGGLVQAGGSLRLSCATSGLTFSVYAMGWFRQAPGKQREFVARITAGGSGTYYADSMEGRFTISRDNARNTVYLQMNSLKPEDTAVYYCAAARHWTRGTEHLPTAYDYWGQGTQVTVSS (SEQ ID NO: 17) PcrV9A (2B7)EVQLVQSGGGLVQAGGSLRLSCASSGSTFRTYGMGWFRQPPGKQREWVAGMAIDGLTTYADSAKGRFTASRDNARNIVYLQMNELKPEDTAVYYCYAAGYWGQGTQVTVSS (SEQ ID NO: 18)PcrV10A (2G6) EVQLVQSGGGLVQAGGSLRLSCTTSGITFSDNAMYWYRQAPGKQRELVASISSGGWTNYADSVKGRFTISRDNVKNTVTLQMNSLEPEDTALYYCRAAPVRGNFIGRVFWGQGTQVTVSS (SEQ ID NO: 19) PcrV11A (4H7)EVQLVQSGGGLVQPGGSLRLSCAAFGSIFTIGTMGWYRQAPGKQRELVATITRGSSTNYADSVKGRFTISIDSAKNTVYLQMNSLKSEDTAVYYCAADRGAVGPAMRVVADWGQGTQVTVSS (SEQ ID NO: 20) PcrV12A (3B7)EVQLVQSGGGLVQAGGSLRLSCAASGSTFSSNAMYWYRQAPGKQRELVASISDGGFTTYYADSVKGRFTISKDNAENTVYLQMNIMKPEDTAVYYCAASISSRVVVHTAQADYWGQGTQVTVSS (SEQ ID NO: 21) PcrV13A (4G2)EVQLVQSGGGLVQPGGSLRLPCAASGSIFTIGTMGWYRQAPGKQRELVATITRGSSTNYADSVKDRFTISRDNAKRTLHLQMNGLKAEDTAVYYCATDLFENSCPLKHDFWGQGTQVTVSS (SEQ ID NO: 22) PcrV14A (4G10)EVQLVQSGGGLVQAGGSLRLSCAASRITFALYVIDWYRQTPESQRELVARIRPEGLAVYADSVKGRFTISRDNGRNTAYLQMNSLQEEDTAVYYCHADPVFTPGRNDYWGQGTQVTVSS (SEQ ID NO: 23) PcrV15A (4G9)EVQLVQSGGGLVQPGESLRLSCAASGSIFSINTMVWYRQVPGKQRELVASITNQGIPHYADSVKGRFTISRENAKNTVNLQMNSLKPEDTAVYVCNAWIRSDGVSPYLNYWGQGTQVTVSS (SEQ ID NO: 24) PerV16A (3B10)EVQLVQSGGGLVQPGGSLGLSCVGSGSISGIHTMGWYRRAPGNQRELIATATSAGITNYSESVKGRFTISRDNAKSTVYLQMSSLKPEDTGVYYCNDVFGRTSWGQGTQVTVSS (SEQ ID NO: 25)PerV17A (3G1) EVQLVQSGGGLVQPGGSLRLSCAASGNIFGGNVMGWYRQAPGKQRELVAGIGSLGRTTYADSVKGRFSISRDNAKNTVYLQMDSLKPEDTAVYYCNVVRLGGPDYWGQGTQVTVSS (SEQ ID NO: 26) PerV18A (3F2)EVQLVQSGGGLVQAGGSLRLSCTTSGNTFSDNAMYWYRQAPGKQREQVASISSGGWTNYADSVKGRFTISRDNVKNTVTLQMDRLEPEDTALYYCRAAPVRGYLIGRVFWGQGTQVTVSS (SEQ ID NO: 27) PerV19A (4E12)EVQLVQSGGGLVQAGGSLRLSCSASGSNSIFNMGWYRQRPGRQRELVALISSGTGSTSYAGSVKGRFAISRDNAKATVYLQMNSLKLEDTAVYYCRITTDNARLVYWGQGTQVTVSS (SEQ ID NO: 28) PerV20A (3C1)EVQLVQSGGGLVQPGGSLRLSCAASGRIFSVNNMGWYRQTPGKQRELVAVITVNGITTYSDSVKGRFTLSRDNAKNTIYLQMNSLKPEDTAVYSCYGYIRLAATNPYVQYWGQGTQVTVSS (SEQ ID NO: 29) PcrV21A (3C7)EVQLVQSGGGLVQPGGSLRLSCAASGSIFSINTMGWYRQAPGNQRDIVATITMNGVPHYADAVKGRFTISRDNAKNTVYLQMNGLKPEDTAVYYCNAWINLYGSPPLQNYWGQGTQVTVSS (SEQ ID NO: 30) PcrV22A (4H8)EVQLVQSGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRELVTSITNQGIPHYADSVKGRFTISRENAKNTVNLQMNSLKPEDTAVYVCNAWIRGDGGSPYLNYWGQGTQVTVSS (SEQ ID NO: 31) PcrV23A (1G6)EVQLVQSGGGLVQPGESLRLSCAASGSIFSINTMVWYRQVPGKQRELVASITNQGIPHYADSVKGRFTISRENAKNTVNLQMNSLKPEDTAVYVCNAWIRSDGVPPYLNYWGQGTQVTVSS (SEQ ID NO: 32) PcrV24A (2E1)EVQLVQSGGGLVQPGGSLRLSCAASGSIFNINSMHWYRQAPGNQRELVASISKGGITNYADSVKGRFAISRDDAQNTLYLQMNSLKPEDTAVYYCNAWISEIATGPILYNYWGQGTQVTVSS (SEQ ID NO: 33) PcrV25A (1F9)EVQLVQSGGGLVQPGGSLSLSCAASGSVFSINRMAWYRQAPGKQRELVADIGTMGASDYADSVKGRFTISRDNAKKTVDLQMNSLKPEDTAVYFCNAWMRGAPDVAYTNYWGQGTQVTVSS (SEQ ID NO: 34) PcrV26A (4H1)EVQLVQSGGGLVQPGGSLRLSCAASGRVVSINNMGWYQQTPGNQRELVAIITLNGVTTYADSVKGRFTISRDNAKNTVYLQMASLKPEDTAIYYCNAWVRTVPGSAYSNYWGQGTQVTVSS (SEQ ID NO: 35) PcrV27A (1B4)EVQLVQSGGDLVQPGGSLRLSCAASGRIFSVNNMGWYRQAPGKQRELVAVITMNGVTTYEDSVKGRFTLSRDNAKNTIYLQMNSLKPEDTAVYFCYGYIRLAATNPYVQYAVGQGTQVTVSS (SEQ ID NO: 36) hzPerV15v1EVQLLESGGGEVQPGGSLRLSCAASGSIFSINTMVWYRQAPGKQRELVSSITNQGIPHYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCNAWIRSDGVSPYLNYWGQGTLVTVKP (SEQ ID NO: 37) hzPcrV15v2EVQLLESGGGEVQPGGSLRLSCAASGSIFSINTMVWYRQAPGKQRELVSSITNQGIPHYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCNAWIRSYGVSPYLNYWGQGTLVTVKP (SEQ ID NO: 38) hzPcrV15v3EVQLLESGGGEVQPGGSLRLSCAASGSIFSINTMVWYRQAPGKQRELVSSITNQGIPHYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCNAWIRSEGVSPYLNYWGQGTLVTVKP (SEQ ID NO: 39) hzPcrV15v4EVQLLESGGGEVQPGGSLRLSCAASGSIFSINTMVWYRQAPGKQRELVSSITNQGIPHYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCNAWIRSQGVSPYLNYWGQGTLVTVKP (SEQ ID NO: 40) hzPerV15DAv5EVQLLESGGGEVQPGGSLRLSCAASGSIFSINTMVWYRQAPGKQRELVSSITNQGIPHYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCNAWIRSDAVSPYLNYWGQGTLVTVKP (SEQ ID NO: 41) hzPerV15DTv6EVQLLESGGGEVQPGGSLRLSCAASGSIFSINTMVWYRQAPGKQRELVSSITNQGIPHYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCNAWIRSDTVSPYLNYWGQGTLVTVKP (SEQ ID NO: 42) hzPcrV15v7EVQLLESGGGEVQPGGSLRLSCAASGSIFSINTMVWYRQAPGKGRELVSSITNQGIPHYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCNAWIRSQGVSPYLNYWGQGTLVTVKP (SEQ ID NO: 81) hzPcrV15v8EVQLLESGGGEVQPGGSLRLSCAASGSIFSINTMVWYRQAPGKGLELVSSITNQGIPHYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCNAWIRSQGVSPYLNYWGQGTLVTVKP (SEQ ID NO: 82) hzP crV18EVQLLESGGGEVQPGGSLRLSCAASGNTFSDNAMYWYRQAPGKQRELVSSISSGGWTNYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCRAAPVRGYLIGRVFWGQGTLVTVKP (SEQ ID NO: 43) hzPerV18v2EVQLLESGGGEVQPGGSLRLSCAASGNTFSDNAMYWYRQAPGKGRELVSSISSGGWTNYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCRAAPVRGYLIGRVFWGQGTLVTVKP (SEQ ID NO: 83) hzPcrV18v3EVQLLESGGGEVQPGGSLRLSCAASGNTFSDNAMYWYRQAPGKGLELVSSISSGGWTNYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCRAAPVRGYLIGRVFWGQGTLVTVKP (SEQ ID NO: 84) hzPcrV20v1EVQLLESGGGEVQPGGSLRLSCAASGRIFSVNNMGWYRQAPGKQRELVSVITVNGITTYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCYGYIRLAATNPYVQYWGQGTLVTVKP (SEQ ID NO: 44) hzPcrV20v2EVQLLESGGGEVQPGGSLRLSCAASGRIFSVNNMGWYRQAPGKQRELVSVITVGGITTYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCYGYIRLAATNPYVQYWGQGTLVTVKP (SEQ ID NO: 71) hzPcrV20v3EVQLLESGGGEVQPGGSLRLSCAASGRIFSVNNMGWYRQAPGKQRELVSVITVQGITTYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCYGYIRLAATNPYVQYWGQGTLVTVKP (SEQ ID NO: 72) hzPcrV20v4EVQLLESGGGEVQPGGSLRLSCAASGRIFSVNNMGWYRQAPGKQRELVSVITNQGITTYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCYGYIRLAATNPYVQYWGQGTLVTVKP (SEQ ID NO: 73) hzPcrV20v5EVQLLESGGGEVQPGGSLRLSCAASGRIFSVNNMGWYRQAPGKQRELVSVITVSGITTYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCYGYIRLAATNPYVQYWGQGTLVTVKP (SEQ ID NO: 74) hzPcrV20v6EVQLLESGGGEVQPGGSLRLSCAASGRIFSVNNMGWYRQAPGKGRELVSVITNQGITTYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCYGYIRLAATNPYVQYWGQGTLVTVKP (SEQ ID NO: 85) hzPcrV20v7EVQLLESGGGEVQPGGSLRLSCAASGRIFSVNNMGWYRQAPGKGLELVSVITNQGITTYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCYGYIRLAATNPYVQYWGQGTLVTVKP (SEQ ID NO: 86) hzPcrV20v8EVQLLESGGGEVQPGGSLRLSCAASGRIFSVNNMGWYRQAPGKGLEWVSVITNQGITTYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCYGYIRLAATNPYVQYWGQGTLVTVKP (SEQ ID NO: 87)

TABLE 2 OprI Binding Protein SequencesOprI-VHH-1 (also referred to herein as ″OprI-1″)QLQLQESGGGLVQSGRSLRLSCSASGSLFRFDTVWWYRQAPGKQREWVAYITAGGMTNYADSVKGRFTISKDNAKNMVYLQMDSLLPEDTAVYYCNVGRNWGQGTQVTVSS (SEQ ID NO: 46)OprI-VHH-2 (also referred to herein as ″OprI-2″)EVQLVQSGGGLVQPGESLRLSCAASGNIFRFDTVWWYRQPPGEQREWVSYITAGSITNYADSVKGRFTISRDNAKNMVYLQMDNLKPEDTAVYYCRVGGSSWGQGTQVTVSS (SEQ ID NO: 47)OprI-VHH-3 (also referred to herein as ″OprI-3″)EVQLVQSGGGLVQAGDSLRLSCAASGGISSTYAMGWFRQAPGKEREFVASIRLGSEATYYADSVKGRFTISRDNALKTIYLQMNSLKPDDTAVYYCAVDASLFLVTVDYWGRGTQVTVSS (SEQ ID NO: 48) OprI-VHH-4 (also referred to herein as ″OprI-4″)QVQLVQSGGGLVQAGGSLRLSCAASGRTFSRCVMGWFRQAPGKEREFVATISWSGASTVYADSVKGRFTISRENAKNTVYLQMNSLKPEDTAVYYCAAAESSWNGDIRLKGYDYWGQGTQVTVSS (SEQ ID NO: 49)OprI-VHH-5 (also referred to herein as ″OprI-5″)QVTLKESGGGLVQAGGSLRLSCAASGRSFRTYTMAWFRQPPGKEREFVAAITWSGGSTFYADPVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYYCAVETSISGRYTVFQPRFYDSWGQGTQVTVSS (SEQ ID NO: 50)OprI-VHH-6 (also referred to herein as ″OprI-6″)QVQLQESGGGLVQPGESLRLSCAASGNIFRFDTVWWYRQPPGEQREWVSYITAGSITNYADSVKGRFIISRDNAKNMVYLQMDNLKPEDTAVYYCRVGGGSWGQGTQVTVSS (SEQ ID NO: 51)OprI-VHH-7 (also referred to herein as ″OprI-7″)EVQLVQSGGGLVQPGGSLRLSCIASGSIFSTKTMGWYRQAPGKQREWVALITTGLSTQYLDSLEGRFTISRDNANNRVFLQMNNLKPEDTGVYYCNVVPGRGATYWGKGTQVTVSS (SEQ ID NO: 52) OprI-VHH-8 (also referred to herein as ″OprI-8″)QLQLQESGGGLVQPGRSLRLSCAGSGSIFRYDTVWWYRQAPGKQREWVAYVTAGGITNYADSVKGRFTISKDNAKNMVYLQMDSLLPEDTAVYYCHVGRNWGQGTQVTVSS (SEQ ID NO: 53)OprI-VHH-9 (also referred to herein as ″OprI-9″)QLQLQESGGGLVQAGGSLRLSCAASGRTFSSNVYSMGWFRQAPGKEREFVSAITWRGGTTYYADSVKDRFTISKDNAKNTVYLQMNSLKSEDTAVYYCACSRMDSTRYDYWGQGTQVTVSS (SEQ ID NO: 54)OprI-VHH-11 (also referred to herein as ″OprI-11″)EVQLVQSGGGLVQSGRSLRLSCSASGSLFRFDTVWWYRQAPGKQREWVAYITAGGITNYADSVKGRFTISKDNAKNMVYLQMDSLLPEDTAVYYCSVGRNWGQGTQVTVSS (SEQ ID NO: 55)OprI-VHH-12 (also referred to herein as ″OprI-12″)QVQLQESGGGLVQPGGSLRLSCAASGITVRINTMGWYRQAPGKQRELVAYITSGGITNYVDSVKGRFTIARDDAKNTVYLQMNSLKPEDTAVYYCNVHGWRDFWGQGTQVTVSS (SEQ ID NO: 56)OprI-VHH-13 (also referred to herein as ″OprI-13″)QVQLVQSGGGLVQPGGSLRLSCAASGTIFRFNTMAWYRQAPGKQREFVAYITWAGMTGYQDSVQDRFTISRDNAKNTVSLQMNNLKPEDTAVYFCNKHGSSFVRDYWGQGTQVTVSS (SEQ ID NO: 57) OprI-VHH-14 (also referred to herein as ″OprI-14″)EVQLVQSGGGLVQPGGSLRLSCAAAGSDFAIGAMGWYRQAPGKQRDFVAHITSGGIPSFADSVKGRFTLSRDNAKNTVYLQMDSLKPDDTAVYYCYLRKRGSGTTTWGQGTQVTVSS (SEQ ID NO: 58) OprI-VHH-15 (also referred to herein as ″OprI-15″)QVQLQESGGGLVQAGGSLRLSCAASGRIFSNCVMGWFRQAPGKEREFVAAISWSGDTTHYADSLKGRFAISRDNANNTVFLQKDSLTPSDTAVYYCAASSRITSCQAMGVVPLLQPWYDYWGRGTQVTVSS (SEQ ID NO: 59)OprI-VHH-16 (also referred to herein as ″OprI-16″)QVQLQESGGGLVQPGRSLRLSCAASGNIFRFDTVWWYRQAPGKQREWVAYVTAGGITNYADSVKGRFTISKDNAKNIVYLHTDNLAPEDTAVYYCRVGQNWGQGTQVTVSS (SEQ ID NO: 60)OprI-VHH-17 (also referred to herein as ″OprI-17″)QLQLQESGGGLVQPGGSPRLSCAASESIFRFNTMAWYRQAPGKQRELVAYITWAGRTDYGDFVKGRFTISRDNAKNTVSLQMNSLKPEDTAVYYCNKHGSRFERDYWGQGTQVTVSS (SEQ ID NO: 61) OprI-VHH-18 (also referred to herein as ″OprI-18″)QVQLQESGGDLVQPGGSLRLSCVASETIFRFNTMAWYRQAPGKRRELVGYITWAGRTGYGDFVEGRFTISRDNSKNTVSLQMNSLKPEDTAVYYCNKHGSSFTQDWGQGTQVTVSS (SEQ ID NO: 62) OprI-VHH-19 (also referred to herein as ″OprI-19″)QLQLQESGGDLVQPGGSLRLSCVASETIFRFNTMAWYRQAPGKRRELVGYITWAGRTGYGDFVEGRFTVSRDNSKNTVSLQMNSLKPEDTAVYYCNKHGASFTQDYWGQGTQVTVSS (SEQ ID NO: 63) OprI-VHH-21 (also referred to herein as ″OprI-21″)QLQLQESGGGLVRPGSSLTLSCVASETIFRFNTMAWYRQAPGKRRELVGYITWAGRTGYGDFVEGRFTISRDNSKNTVSLQMNSLEPEDTADYYCNKHGSSFLRDYWGQGTQVTVSS (SEQ ID NO: 64) OprI-VHH-22 (also referred to herein as ″OprI-22″)QVQLQESGGGLVQPGRSLRLSCAGSGSMFRFDTVWWYRQAPGKQRDWVSYITAGSIANYADSVKGRFTISRDNTKNMVYLQMDSLKPEDTAVYYCRVGGNSWGQGTQVTVSS (SEQ ID NO: 65)OprI-VHH-23 (also referred to herein as ″OprI-23″)QVQLQQSGGGLVQPGGSLRLSCEASSNIFRFNTMAWYRQAPGKQREFAAYITWAGLTGYGDSLKGRFIISRDNAKNIVTLQMNSLKPEDTAVYYCNKHGSDFVRDYWGQGTQVTVSS (SEQ ID NO: 66) hzOprI-7v1 (also referred to herein as ″OprI-7v1″)EVQLLESGGGEVQPGGSLRLSCAASGSIFSTKTMGWYRQAPGKQREWVSLITTGLSTQYAESVKGRFTISRDNANNTVYLQMSSLRAEDTAVYYCNVVPGRGATYWGQGTLVTVKP (SEQ ID NO: 67) hzOprI-7v2 (also referred to herein as ″OprI-7v2″)EVQLLESGGGEVQPGGSLRLSCAASGSIFSTKTMGWYRQAPGKQREWVSLITTGLSTQYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCNVVPGRGATYWGQGTLVTVKP (SEQ ID NO: 68) hzOprIv3 (also referred to herein as ″OprI-7v3″)EVQLLESGGGEVQPGGSLRLSCAASGSIFSTKTMGWYRQAPGKGLEWVSLITTGLSTQYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCNVVPGRGATYWGQGTLVTVKP (SEQ ID NO: 69) hzOprI-7v4 (also referred to herein as ″OprI-7v4″)EVQLLESGGGEVQPGGSLRLSCAASGSIFSTKTMGWYRQAPGKGLEWVSLITTGLSTQYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCNVVPGRGATYWGQGTLVTVKP (SEQ ID NO: 70) hzOprI-7v5EVQLLESGGGEVQPGGSLRLSCAASGSIFSTKTMGWYRQAPGKGREWVSLITTGLSTQYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCNVVPGRGATYWGQGTLVTVKP (SEQ ID NO: 88)

1.-43. (canceled)
 44. A method for treating an infectious disease, themethod comprising administering to a subject in need thereof atherapeutically effective amount of an isolated polypeptide comprising afirst VHH domain that binds PcrV from Pseudomonas aeruginosa, whereinthe first VHH domain comprises the CDRs of SEQ ID NO: 41, as determinedby Chothia numbering.
 45. The method of claim 44, wherein the first VHHdomain is humanized.
 46. The method of claim 44, wherein the polypeptidecomprises an immunoglobulin Fc region polypeptide.
 47. The method ofclaim 46, wherein the immunoglobulin Fc region polypeptide comprises theamino acid sequence of SEQ ID NO: 1, 2, 3, or
 4. 48. The method of claim44, wherein the infectious disease is a Pseudomonas aeruginosainfection.
 49. The method of claim 48, wherein the Pseudomonasaeruginosa infection is a community acquired infection.
 50. The methodof claim 48, wherein the Pseudomonas aeruginosa infection is anosocomial infection.
 51. The method of claim 44, wherein thepolypeptide comprises a second VHH domain that binds OprI fromPseudomonas aeruginosa.
 52. The method of claim 51, wherein the secondVHH domain comprises the CDRs of SEQ ID NO: 62, as determined by Chothianumbering.
 53. The method of claim 52, wherein the first VHH domain andthe second VHH domain are humanized.
 54. The method of claim 52, whereinthe polypeptide comprises an immunoglobulin Fc region polypeptide. 55.The method of claim 54, wherein the immunoglobulin Fc region polypeptidecomprises the amino acid sequence of SEQ ID NO: 1, 2, 3, or
 4. 56. Themethod of claim 51, wherein the infectious disease is a Pseudomonasaeruginosa infection.
 57. The method of claim 56, wherein thePseudomonas aeruginosa infection is a community acquired infection. 58.The method of claim 56, wherein the Pseudomonas aeruginosa infection isa nosocomial infection.
 59. The method of claim 52, wherein thepolypeptide comprises a third VHH domain that binds PcrV fromPseudomonas aeruginosa, wherein the first VHH domain and the third VHHdomain bind different epitopes of PcrV.
 60. The method of claim 59,wherein the polypeptide comprises an immunoglobulin Fc regionpolypeptide.
 61. The method of claim 60, wherein the immunoglobulin Fcregion polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2,3, or
 4. 62. The method of claim 59, wherein the infectious disease is aPseudomonas aeruginosa infection.
 63. The method of claim 62, whereinthe Pseudomonas aeruginosa infection is a community acquired infection.64. The method of claim 62, wherein the Pseudomonas aeruginosa infectionis a nosocomial infection.
 65. A method for treating an infectiousdisease, the method comprising administering to a subject in needthereof a therapeutically effective amount of an isolated polypeptidecomprising a first VHH domain that binds OprI from Pseudomonasaeruginosa, wherein the first VHH domain comprises the CDRs of SEQ IDNO: 62, as determined by Chothia numbering.
 66. The method of claim 65,wherein the first VHH domain is humanized.
 67. The method of claim 65,wherein the polypeptide comprises an immunoglobulin Fc regionpolypeptide.
 68. The method of claim 67, wherein the immunoglobulin Fcregion polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2,3, or
 4. 69. The method of claim 65, wherein the polypeptide comprises asecond VHH domain that binds PcrV from Pseudomonas aeruginosa.
 70. Themethod of claim 69, wherein the polypeptide comprises an immunoglobulinFc region polypeptide.
 71. The method of claim 70, wherein theimmunoglobulin Fc region polypeptide comprises the amino acid sequenceof SEQ ID NO: 1, 2, 3, or
 4. 72. The method of claim 65, wherein theinfectious disease is a Pseudomonas aeruginosa infection.
 73. The methodof claim 72, wherein the Pseudomonas aeruginosa infection is a communityacquired infection.
 74. The method of claim 72, wherein the Pseudomonasaeruginosa infection is a nosocomial infection.
 75. A method forpreventing an infectious disease, the method comprising administering toa subject at risk of developing an infectious disease an effectiveamount of an isolated polypeptide comprising a first VHH domain thatbinds PcrV from Pseudomonas aeruginosa, wherein the first VHH domaincomprises the CDRs of SEQ ID NO: 41, as determined by Chothia numbering.76. A method for preventing an infectious disease, the method comprisingadministering to a subject at risk of developing an infectious diseasean effective amount of an isolated polypeptide comprising a first VHHdomain that binds OprI from Pseudomonas aeruginosa, wherein the firstVHH domain comprises the CDRs of SEQ ID NO: 62, as determined by Chothianumbering.